PDK1 mediates activation of PKB, p70 S6 kinase and p90 Rsk in vivo, but is not rate-limiting for activation of PKA, MSK1 and AMPK. Another kinase phosphorylates PKB at its hydrophobic motif in PDK1(-/-) cells. PDK1 phosphorylates the hydrophobic motif of p70 S6 kinase either directly or by activation of another kinase.

PDK1 and PDK2 might be the same enzyme, the substrate specificity and activity of PDK1 being regulated through its interaction with another protein(s). PRK2 is a probable substrate for PDK1.

The activation of phosphoinositide 3-kinase (PI3K) by insulin represents a key signalling event in the hormonal stimulation of diverse cellular responses including glucose transport and glycogen synthesis. The activation of PI3K increases the production of 4,5 trisphosphate [PtdIns(3,4,5)P 3 ] and phosphatidylinositol 3,4 bisphosphate [PtdIns(3,4)P 2 ]), which act as important signalling intermediates in the downstream activation of the serine/threonine kinase, Protein Kinase B (PKB/Akt). Activation of PKB depends upon its phosphorylation on two key amino acid residues, Thr 308 and Ser 473, with full activation requiring the phosphorylation of both [1]. The Nterminal domain of PKB contains a pleckstrin homology (PH) domain, which is thought to be critical in allowing the kinase to interact with 3-phospho- Diabetologia (2001) Abstract Aims/hypothesis. Increased cellular production of ceramide has been implicated in the pathogenesis of insulin resistance and in the impaired utilisation of glucose. In this study we have used L6 muscle cells to investigate the mechanism by which the short-chain ceramide analogue, C 2 -ceramide, promotes a loss in insulin sensitivity leading to a reduction in insulin stimulated glucose transport and glycogen synthesis. Method. L6 muscle cells were pre-incubated with C 2 -ceramide and the effects of insulin on glucose transport, glycogen synthesis and the activities of key molecules involved in proximal insulin signalling determined.Results. Incubation of L6 muscle cells with ceramide (100 mmol/l) for 2 h led to a complete loss of insulinstimulated glucose transport and glycogen synthesis. This inhibition was not due to impaired insulin receptor substrate 1 phosphorylation or a loss in phosphoinositide 3-kinase activation but was caused by a failure to activate protein kinase B. This defect could not be attributed to inhibition of 3-phosphoinositidedependent kinase-1, or to impaired binding of phosphatidylinositol 3,4,5 triphosphate (PtdIns(3,4,5)P 3 ) to the PH domain of protein kinase B, but results from the inability to recruit protein kinase B to the plasma membrane. Expression of a membrane-targetted protein kinase B led to its constitutive activation and an increase in glucose transport that was not inhibited by ceramide. Conclusions/interpretation. These findings suggest that a defect in protein kinase B recruitment underpins the ceramide-induced loss in insulin sensitivity of key cell responses such as glucose transport and glycogen synthesis in L6 cells. They also suggest that a stimulated rise in PtdIns(3,4,5)P 3 is necessary but not sufficient for protein kinase B activation in this system. [Diabetologia (2001) 44: 173±183]

Members of the AGC subfamily of protein kinases including protein kinase B, p70 S6 kinase, and protein kinase C (PKC) isoforms are activated and/or stabilized by phosphorylation of two residues, one that resides in the T-loop of the kinase domain and the other that is located C-terminal to the kinase domain in a region known as the hydrophobic motif. Atypical PKC isoforms, such as PKC, and the PKC-related kinases, like PRK2, are also activated by phosphorylation of their T-loop site but, instead of possessing a phosphorylatable Ser/Thr in their hydrophobic motif, contain an acidic residue. The 3-phosphoinositide-dependent protein kinase (PDK1) activates many members of the AGC subfamily of kinases in vitro, including PKC and PRK2 by phosphorylating the T-loop residue. In the present study we demonstrate that the hydrophobic motifs of PKC and PKC, as well as PRK1 and PRK2, interact with the kinase domain of PDK1. Mutation of the conserved residues of the hydrophobic motif of full-length PKC, fulllength PRK2, or PRK2 lacking its N-terminal regulatory domain abolishes or significantly reduces the ability of these kinases to interact with PDK1 and to become phosphorylated at their T-loop sites in vivo. Furthermore, overexpression of the hydrophobic motif of PRK2 in cells prevents the T-loop phosphorylation and thus inhibits the activation of PRK2 and PKC. These findings indicate that the hydrophobic motif of PRK2 and PKC acts as a "docking site" enabling the recruitment of PDK1 to these substrates. This is essential for their phosphorylation by PDK1 in cells.

The multi-site phosphorylation of the protein kinase C (PKC) superfamily plays an important role in the regulation of these enzymes. One of the key phosphorylation sites required for the activation of all PKC isoforms lies in the T-loop of the kinase domain. Recent in vitro and transfection experiments indicate that phosphorylation of this residue can be mediated by the 3-phosphoinositide-dependent protein kinase-1 (

-473 as well as Thr-308. This suggests that PDK1 may be the enzyme that phosphorylates both residues in vivo. Here we demonstrate that PDK1 is capable of phosphorylating p70 S6 kinase at Thr-412 in vitro. We study the effect of PIF on the ability of PDK1 to phosphorylate p70 S6 kinase. Surprisingly, we find that PDK1 bound to PIF is no longer able to interact with or phosphorylate p70 S6 kinase in vitro at either Thr-252 or Thr-412. The expression of PIF in cells prevents insulin-like growth factor 1 from inducing the activation of the p70 S6 kinase and its phosphorylation at Thr-412. Overexpression of PDK1 in cells induces the phosphorylation of p70 S6 kinase at Thr-412 in unstimulated cells, and a catalytically inactive mutant of PDK1 prevents the phosphorylation of p70 S6K at Thr-412 in insulin-like growth factor 1-stimulated cells. These observations indicate that PDK1 regulates the activation of p70 S6 kinase and provides evidence that PDK1 mediates the phosphorylation of p70 S6 kinase at Thr-412. p70 S6 kinase (p70 S6K)1 is activated by insulin and growth factors and mediates the phosphorylation of the 40 S ribosomal protein S6 (1). This enables efficient translation of mRNA molecules containing a polypyrimidine tract at their 5Ј-transcriptional start sites (2). p70 S6K also phosphorylates unknown proteins to permit progression through the G 1 phase of the cell cycle (3). p70 S6K is activated by insulin and growth factors, through a phosphoinositide 3-kinase-dependent pathway and becomes phosphorylated on at least 7 Ser/Thr residues in response to these agonists. The phosphorylation of two of these residues namely Thr-252 and Thr-412 on the longer splice variant of the ␣-isoform (Thr-229 and Thr-389 on the shorter splice variant) appears to make the most important contribution to the activation of p70 S6K (4 -6). Phosphorylation of Thr-252 alone or mutation of Thr-412 to glutamic acid to mimic phosphorylation of this residue results in a small activation of p70 S6K. In contrast, phosphorylation of both residues or phosphorylation of Thr-252 in the T412E mutant of p70 S6K results in a large activation of expressed p70 S6K, showing that phosphorylation of Thr-252 and Thr-412 leads to a synergistic activation p70 S6K (7,8).p70 S6K is a member of the AGC subfamily of protein kinases, which include protein kinase B (PKB) (9), protein kinase C (PKC) isoforms (10), and serum-and glucocorticoid-regulated protein kinase (11). The residues surrounding Thr-252 and Thr-412 of p70 S6K are highly conserved in all AGC family members, and phosphorylation of the residues equivalent to Thr-252 and Thr-412 of p70 S6K is necessary for activation and/or stability of these kinases in vivo (12). Thr-252 is located between subdomains VII and VIII of the kinase domain, a region whose phosphorylation activates many kinases. The residue equivalent to Thr-252 lies in a Thr-Phe-Cys-Gly-ThrXaa-Glu-Tyr consensus motif (where the underlined Thr corresponds to Thr-252 and Xaa is a variable residue). Thr-412 is located C-terminal to the ca...

Obesity is associated with chronic low-grade inflammation that contributes to defects in energy metabolism and insulin resistance. Suppressor of cytokine signaling (SOCS)-3 expression is increased in skeletal muscle of obese humans. SOCS3 inhibits leptin signaling in the hypothalamus and insulin signal transduction in adipose tissue and the liver. Skeletal muscle is an important tissue for controlling energy expenditure and whole-body insulin sensitivity; however, the physiological importance of SOCS3 in this tissue has not been examined. Therefore, we generated mice that had SOCS3 specifically deleted in skeletal muscle (SOCS MKO). The SOCS3 MKO mice had normal muscle development, body mass, adiposity, appetite, and energy expenditure compared with wild-type (WT) littermates. Despite similar degrees of obesity when fed a high-fat diet, SOCS3 MKO mice were protected against the development of hyperinsulinemia and insulin resistance because of enhanced skeletal muscle insulin receptor substrate 1 (IRS1) and Akt phosphorylation that resulted in increased skeletal muscle glucose uptake. These data indicate that skeletal muscle SOCS3 does not play a critical role in regulating muscle development or energy expenditure, but it is an important contributing factor for inhibiting insulin sensitivity in obesity. Therapies aimed at inhibiting SOCS3 in skeletal muscle may be effective in reversing obesity-related glucose intolerance and insulin resistance.

In this study we show that serotonin (5-hydroxytryptamine (5-HT)) causes a rapid stimulation in glucose uptake by ϳ50% in both L6 myotubes and isolated rat skeletal muscle. This activation is mediated via the 5-HT 2A receptor, which is expressed in L6, rat, and human skeletal muscle. In L6 cells, expression of the 5-HT 2A receptor is developmentally regulated based on the finding that receptor abundance increases by over 3-fold during differentiation from myoblasts to myotubes. Stimulation of the 5-HT 2A receptor using methylserotonin (m-HT), a selective 5-HT 2A agonist, increased muscle glucose uptake in a manner similar to that seen in response to 5-HT. The agonist-mediated stimulation in glucose uptake was attributable to an increase in the plasma membrane content of GLUT1, GLUT3, and GLUT4. The stimulatory effects of 5-HT and m-HT were suppressed in the presence of submicromolar concentrations of ketanserin (a selective 5-HT 2A antagonist) providing further evidence that the increase in glucose uptake was specifically mediated via the 5-HT 2A receptor. Treatment of L6 cells with insulin resulted in tyrosine phosphorylation of IRS1, increased cellular production of phosphatidylinositol 3,4,5-phosphate and a 41-fold activation in protein kinase B (PKB/Akt) activity. In contrast, m-HT did not modulate IRS1, phosphoinositide 3-kinase, or PKB activity. The present results indicate that rat and human skeletal muscle both express the 5-HT 2A receptor and that 5-HT and specific 5-HT 2A agonists can rapidly stimulate glucose uptake in skeletal muscle by a mechanism which does not depend upon components that participate in the insulin signaling pathway.Serotonin, also known as 5-hydroxytryptamine (5-HT), 1 is a neurotransmitter that has been implicated in the regulation of diverse physiological processes, including cellular growth and differentiation (1), neuronal development (2), and regulation of blood glucose concentration (3,4). This functional diversity stems from the ability of the neurotransmitter to interact with multiple 5-HT receptors (currently classified as 5-HT 1 through to 5-HT 7 with further subtypes within each receptor class) that can trigger and activate distinct intracellular signaling systems (5). For example, with the exception of the 5-HT 3 receptor which operates as a ligand-gated ion channel, all known 5-HT receptors belong to the superfamily of G-protein coupled receptors which can, depending on receptor class and subtype, couple negatively or positively to adenylyl cyclase (6), modulate ion channel activity (7), promote hydrolysis of phosphatidylinositol bisphosphate through activation of phospholipase C-␤ (8, 9), and stimulate the mitogen-activated protein kinase pathway (10).Of major interest to us, however, has been the observation that administration of 5-HT, 5-HT precursors, or specific 5-HT receptor agonists and antagonists can modulate circulating levels of blood glucose in rodents. While some investigators have reported that 5-HT promotes hyperglycemia by a mechanism that may involve ...